Project Summary Drug delivery across the blood brain barrier is a significant challenge in developing successful therapeutics for brain malignancies. Despite significant advances in developing small molecules, peptides, and nucleic acid treatment options, successful delivery of these molecules to the brain diminishes their promise. We propose a novel approach of delivering small interfering RNA (siRNA) across the blood brain barrier for therapeutic applications, harnessing the body?s natural microRNA delivery vehicle: high density lipoproteins. HDL particles bind specifically to the scavenger receptor class B type 1 (SR-B1) and unload their core contents directly to the cytosol of cells?bypassing the endo-lysosomal pathway?which facilitates delivery of cholesterol and phospholipids to steroidogenic tissues and the liver.36,37 Our chief scientific collaborator has developed unique adaptations of this natural lipid delivery system to deliver diverse drugs and therapeutic agents, including therapeutic nucleic acids, via reconstituted HDL (rHDL) nanoparticles?particles with specified phospholipid and apolipoprotein content analogous to native HDL, demonstrating their ability to enter the brain.38?40 As an advantage over small molecules, this lipoprotein formulation has a long circulating half-life, delivers therapeutic payloads directly to the cytosol, and targets the SR-B1 receptor, which is expressed on the BBB and enables transcytosis of the particle into the brain. While our prior data demonstrating proof-of-concept rHDL- mediated siRNA delivery is in SR-B1-overexpressing ovarian cancer cells, there is strong evidence in the literature to support our proposal for rHDL particles crossing the BBB for efficient therapeutic siRNA delivery. As an initial proof-of-concept study, we will target asparagine endopeptidase (AEP)?an enzyme recently discovered to cleave both APP and tau, the two key components of Alzheimer?s Disease pathology. AEP has been demonstrated to be activated and elevated in human AD brains leading to increasedA? and tau accumulation, and progressive neurodegeneration. A novel small molecule AEP inhibitor was recently developed that reduces tau and APP cleavage, improves long-term potentiation, and improves memory protection in transgenic AD mice upon oral administration. While these pre-clinical results are encouraging, the safety and specificity have yet to be determined and this small molecule will likely face typical challenges for drug delivery to the brain: short residence time in circulation and low blood-brain barrier (BBB) permeability. Thus, there exists an urgent need to develop specific and effective AEP inhibition for AD treatment. The focus of this proposal is to accomplish key milestones that will establish proof-of-concept BBB-crossing drug delivery to the brain. We aim to design, screen, and select a novel AEP-siRNA for AD treatment. Delivery will be mediated via rHDL to determine biodistribution and measure AEP gene silencing effects in an AD mouse model.